Rong Guang-hua, Xia Yi, Zhu Shi-ying, Tong Yi-min, Qi Zhong-tian
Department of Microbiology, Key Laboratory for Medical Microbiology, Second Military Medical University, Shanghai 200433, China.
Wei Sheng Wu Xue Bao. 2006 Dec;46(6):900-5.
A total amount of 117 candidate chromosomal sequences were screened for the genomic signatures of Bacillus anthracis by a 2-step approach. Out of them, 19 genomic signatures sequences were selected, among which, 6 genomic signatures were competent as the target sequence of TaqMan real-time PCR method. With the most significant alignment and specificity, fragment C04, together with pagA gene and capB gene from virulence plasmids pX01 and pX02, was selected to establish a TaqMan real-time PCR assay. For each target sequence, as little as 10 to 100 copies per reaction could be detected. Twelve bacterial species including 7 from Bacillus cereus group which were closely related to Bacillus anthracis were used to test the specificity of this assay. Data revealed that the assay gained a 100% specificity. Further performance of the assay was assessed with 10 simulative contaminated environmental samples and 20 negative control environmental samples; all of the Bacillus anthracis contaminated samples gave strong positive signals while the control samples were negative. This specific and sensitive real-time PCR assay could be used in rapid confirmation or exclusion of potential bio-attacks and the diagnosis of anthrax.
采用两步法对117条候选染色体序列进行炭疽芽孢杆菌基因组特征筛选。从中筛选出19条基因组特征序列，其中6条基因组特征可作为TaqMan实时荧光定量PCR方法的靶序列。经比对和特异性分析，选取片段C04，联合来自毒力质粒pX01和pX02的pagA基因及capB基因，建立TaqMan实时荧光定量PCR检测方法。对于每个靶序列，每个反应可检测低至10至100个拷贝。选取包括7种与炭疽芽孢杆菌密切相关的蜡样芽孢杆菌属细菌在内的12种细菌，对该检测方法的特异性进行检测。结果显示，该检测方法特异性达100%。进一步使用10份模拟污染环境样本和20份阴性对照环境样本对该检测方法进行性能评估；所有炭疽芽孢杆菌污染样本均给出强阳性信号，而对照样本均为阴性。这种特异性强、灵敏度高的实时荧光定量PCR检测方法可用于快速确认或排除潜在的生物攻击以及炭疽的诊断。
